This application is based upon the hypothesis that intracellular antioxidant status is an important component of the endothelial cell dysfunction that characterizes atherosclerosis and contributes to the clinical manifestations of atherosclerotic vascular disease. Normally, the endothelium maintains vascular homeostasis, in part, through the action of endothelium-derived nitric oxide (EDNO). There is now compelling evidence that EDNO action is particularly sensitive to increased vascular oxidative stress (an imbalance between oxidants and antioxidants in favor of the former). Until now, research into the mechanism(s) of oxidative stress and impaired EDNO action has primarily involved antioxidant enzymes such as superoxide dismutase or lipid- soluble antioxidants such as alpha-tocopherol, beta-carotene, or probucol. The potential role of water-soluble antioxidants in modulating EDNO action is largely unknown. The goal of this proposal, therefore, is to define to role(s) of vitamin C and glutathione (GSH), the two principal intracellular water-soluble antioxidant species in vivo, on endothelial function. The primary experimental model for this proposal will be human aortic endothelial cells (HAECs) and our assessment of endothelial function will be the action of EDNO. In order to accomplish the goal of this project, we will need to consider the dual roles of vitamin C and GSH within the cell. Both vitamin C and GSH provide reducing equivalents for critical cellular functions and they are important for cellular antioxidant protection in the face of oxidative stress. We will first test the role of vitamin C and GSH in EDNO action and production using unstressed HAECs. We will determine the dependence of EDNO action on the cellular content and redox state of vitamin C and GSH. Once this dependence is established, we will investigate candidate mechanisms including nitric oxide synthase activity, cofactor availability, and NO inactivation by superoxide. The role of vitamin C and GSH will also be tested in HAECs exposed to physiologically relevant sources of oxidative stress such as superoxide, peroxides, oxidized LDL, or native LDL. Finally, we will investigate the roles of vitamin C and GSH on EDNO action in vivo using a guinea pig model. Guinea pigs will be fed diets designed to render them marginally vitamin C deficient or vitamin C adequate. GSH status will also be modulated with buthionine sulfoximine and/or nitrofurantoin. The implications of these manipulations for EDNO action will be determined using EDNO bioassay and related to markers of oxidative stress. As a physiologic source of oxidative stress, these studies will also be performed in the setting of cholesterol feeding which is known to increase vascular oxidative stress through excess vascular superoxide. These studies should provide additional insight into the role(s) of vitamin C and GSH in maintaining vascular homeostasis and may suggest treatment strategies for patients with atherosclerotic vascular disease.